In 1773 O. F. Muller became the first man to train the newly invented microscope
on a dinoflagellate. Yet as early as 77 A.D. the Greek naturalist, Pliny,
in a book of notes on his observations wrote, "There are sudden fires
in the waters," most likely referring to what we now recognize as dinoflagellate
bioluminescence (Nightingale, 1936). Dinoflagellates remained mysterious
until F. Stein published his dinoflagellate monographs in 1878-1883, which
provided the first good morphological description of the creatures. The
brilliantly drawn figures show enough detail to allow modern taxonomists
to classify the species Stein analyzed. To this day, many genera carry the
name originally coined by Stein in his famous monographs. Since then, the
list of identified dinoflagellates has exploded -- in just the last ten
years the number of identified species has doubled -- and our understanding
of the vital role they play has exploded with it. Still, much remains to
be discovered.

Morphologically, dinoflagellates consist of an epitheca and a hypotheca,
separated by a transverse groove, the cingulum. Two flagella allow the single-celled,
eukaryotic organism to propel itself through the water at speeds of up to
370mm/sec. (Spector, 1984). A longitudinal cleft called the sulcul groove,
or sulcum, separates dorsal and ventral surfaces. The spiraling longitudinal
flagellum inserts at the hypothecal end of the sulcum. The apex then denotes
the opposite side. The second flagellum, the transverse flagellum, skirts
the cingular groove like a ribbon.

Theca
Some species protect themselves with a plated covering called the theca, while
others, called "naked" dinoflagellates, have a simple membranous
cuticle. On the ultrastructural level the theca is composed of three membrane
layers which can be either filled with cellulose or unfilled to lend different
levels of stiffness to different species. The number of plates and their
arrangement also varies across species and can be taxonomically useful. Unfortunately
for the taxonomists morphologies can change drastically even within the same
individual depending on environmental circumstances, degree of resolution,
and life stage.

Internal morphology
Internally, dinoflagellates universally possess chlorophylls a and c (though
some may possess different dimers of chlorophyll c with chlorophyll
c2 predominating, and one report claimed that certain dinoflagellates may
possess chlorophyll b and even phycobiliproteins). Like most algae,
their adaption to the low intensity and filtered light that penetrates the
sea comes from their accessory pigments -- mainly carotenes and xanthophylls,
like peridinin and ß-carotene. They also possess various sterols (like
cholesterol). Starch granules in the cytoplasm provide food storage along with
long-chain fatty acids. They possess golgi bodies and vesicles, everything
typifying a good eukaryote, except for the nucleus. A permanent nuclear envelope
contains condensed, banded chromosomes, which is normal, but their DNA lacks
histones and nucleosomes, giving them a "mesokaryotic" nucleus. Like
the external morphology, the kinds and relative amounts of internal structures
varies from species to species, and again may be different at different times
even in the same individual.

Problems
Almost every taxanomically significant aspect in the dinoflagellates varies
remarkably across species and genera, making identification highly labor
and equipment intensive. New technologies are being developed to make the
process easier. One new technique for cyst detection involves fluorescence
tagging with primuline after treatment with methane(Yamaguchi, Itakura, Imai,
and Ishida, 1995). Other taxonomic characteristics include lifestyle (parasitic
vs. symbiotic vs. free living), life history, ultrastructure, and biochemistry.
Even with highly sophisticated analyzing equipment, taxonomic classification
can be a bear. This becomes especially problematic when trying to differentiate
toxic or otherwise ecologically significant species from identical, yet less "important" counterparts.
These problems, coupled with a poor fossil record, have made the story of
how and when dinoflagellates evolved indecipherable. Speculations cover the
full range of possibilities, and the story will remain a mystery until dinoflagellates
appear in older fossil records, or until biochemical assays become more reliable.

Ceratium
In the Monterey Bay, dinoflagellate populations fluctuate seasonally. In a
plankton tow taken from the Hopkins Marine Reserve (mesh size unknown) in
mid-March, the majority of the dinoflagellates observed belonged to the phylum
Ceratium, a thecate dinoflagellate with two long hypothecal horns extending
apically, and one apical horn (see "morphology").
Most of the specimens observed in the tow may even belong to the same species.
The homogeneity of the sample could have several causes. The species recovered
measured about 175µm across and 575µm to the end of its long
apical horn (unpublished data). Most species run from 10µm to 50µm,
according to the literature, so a large mesh may have failed to trap the
smaller species. However, very small diatoms, 20µm in diameter, appeared
in the sample, suggesting that small mesh size was not a problem. Another
cause may have been poor equipment, unable to resolve the smaller dinoflagellates.
A third, and perhaps the most likely cause, requires a little background
information.

Around the world, from polar to tropical seas, people have recorded a near-shore
phenomenon known as a red tide. Darwin noted a red tide off the coast of
South America in 1871 (Nightingale, 1936), and the number and range of reported
red tides has increased over the last hundred years, simply due to an increasing
awareness. The red colored cloud in the water near shore that characterizes
a red tide comes from the aggregation of thousands of dinoflagellates. In
one red tide in Olympia, Washington 15,834 individual cells jammed themselves
into one milliliter of sea water (Nightingale, 1936). The number of different
species of dinoflagellates in an aggregation during a red tide tends to
be minimal, suggesting competition among different dinoflagellate species.
Certain dinoflagellates secrete ectocrines, hormones that subdue other dinoflagellate
species. Dinoflagellates often phagocytize other dinoflagellates. Many factors
determine why one species dominates, and the relationships lack resolution
as of yet, but the appearance of one predominant species in a net tow should
not be too surprising.

PSP
Worldwide attention was drawn to the red tide phenomenon when scientists linked
the occurrence of red tides to another phenomenon known as paralytic shellfish
poisoning (PSP), which has claimed human lives along with birds and other
shellfish feeding animals. Since the discovery of toxic dinoflagellates,
many different species with many different toxins have been characterized.
To date, sixty toxin containing species have been described of the 2,000
extant species (Spector, 1984). Seven years ago taxonomists had described
half as many toxic and non-toxic species.

Other toxins
Vast efforts have gone into this field since the discovery of PSP, yielding
full characterization of about twenty toxins and their physiological effects.
Besides PSP, cases of diarrhetic shellfish poisoning (DSP) and neurotoxic
shellfish poisoning (NSP), as well as fish toxins have been ascribed to dinoflagellates.
Toxins can be neurotoxic, hemolytic, or gastrointestinal in their physiological
effect, and several are potentially lethal.

Saxitoxin
By far the most potent is the toxin implicated in paralytic shellfish poisoning.
In California a rare episode of PSP killed several people and scared the
California Department of Health Services (DHS) into placing an annual quarantine
on shellfish as a protective measure. The toxin is called saxitoxin and acts
by blocking sodium channels. This inhibits neuron depolarizations and action
potentials, leading to respiratory failure. Thirteen derivatives of saxitoxin
have been chemically resolved, and shellfish metabolism may cause interconversion
between different forms of saxitoxin. This could convert a less potent toxin
to lethal one. Shellfish also accumulate and concentrate the toxin, by filtering
out the toxic dinoflagellates as they feed. PSP takes effect in as little
as a half an hour depending on the species consumed and the concentration
consumed (Spector, 1984). In Canada, in 1948, two four year old children
ate toxic clams at the beach. They both died within minutes (Anderson, White,
Baden). Despite intensive study, no anti-dote exists.

Toxin evolution
It is tempting to ask what the selection pressure might be for toxicity in
a single-celled organism. Since even the biochemical pathway for toxin synthesis
is unknown, it is difficult to speculate, but one interesting theory has
been put forth. This theory suggests that the toxin may actually be used
by the dinoflagellate for nitrogen storage. Chemically, saxitoxinoids are
well suited for nitrogen storage, and the cyst form of most dinoflagellates
seems to lack a nitrogen source (Spector, 1984). In this case, the toxicity
of saxitoxin is incidental -- an unfortunate side effect. However, until
more detail is uncovered about how the toxin is made and used by a dinoflagellate,
the theory remains speculative.

Between blooms, the number of dinoflagellates found in the plankton can
be exceedingly low. What prevents blooms from occurring? Why do blooms seem
to occur according to a seasonal schedule? Grazing by copepods and nutrient
limitation accounts for part of this, but blooms occur equally across nutrient
and light gradients. The missing factor responsible for blooms is the cyst
reservoir. The dinoflagellate cyst is an immotile stage in their life history.
Dinoflagellates spend the majority of their life in a haploid, vegetative
state, reproducing asexually by cellular mitosis -- one dinoflagellate pinches
off to form two. (Thecate species have to deal with re-formation of the
theca, and different species use different methods, which taxonomists can
use for classification.) Given an environmental cue, two of the newly formed
dinoflagellates may fuse as gametes to form a "planozygote," which
retains both nuclei and both longitudinal flagella. Various laboratories
have investigated the environmental cues, and while low nitrogen is used
almost universally in vitro for beginning the sexual cycle, one report indicated
that all of the following may induce fusion under laboratory conditions:
low nitrogen, low phosphorous, low light intensity, shortened day length,
decreased water temperature, and increased salinity (Faust, 1992). Most
likely a combination of these act as the environmental cues in vivo, and
all of these are symptomatic of winter in the temperate seas. The planozygote
continues the cycle with a series of morphological changes to become the
cyst, or hypnozygote. It loses all flagella and grows in size, often thickening
its theca by depositing cellulose between thecal membrane layers. Metabolically
active cytoplasm is reduced and replaced with starch granules. In the nucleus,
protective proteins congregate around the chromosomes (Xiaoping, Dodge,
and Lewis, 1989). Presumably these changes prepare the dinoflagellate to
overwinter by slowing metabolism, augmenting food storage, and protecting
against agitation. When they lose their motility, they sink, and it has
been shown in vitro that a resting cyst under ideal conditions may remain
viable for five-and-a-half years (Anderson, White, and Baden, 1985). Cysts
build up in the sediment over the years to form a "cyst reservoir" from
which new dinoflagellates are recruited to initiate a bloom. A temperature
rise induces ecdysis, or excystment, and the hypnozygote reverts to the
vegetative state, reforming both flagella. Where in the cycle meiosis occurs
appears unclear. Indeed it may vary from species to species, and imaging
of the resting cyst has proven difficult due to the thickened theca. But
when the excysted cell emerges it is haploid, like the initial vegetative
cell. Putting everything together, the vegetative cells gradually sink out
of the plankton with the onset of fall, (or generally poor living conditions)
leaving a limited number of free-floating individuals in the plankton. Grazing
rates or nutrient limitation keep the population from exploding at this
point. Then as spring sets in and temperatures increase, hypnozygotes excyst
and new dinoflagellates are recruited. The cyst reservoir contains a huge
number of hypnozygotes -- residuals from not just the previous winter, but
from as many as five seasons past. Given the right conditions -- high nutrient
level, high light level, low grazing, and low competition from other bloom
species -- cysts ecdyze by the thousands. With a recorded growth rate of
as much as one division per day, it only takes 12.5 cysts per square centimeter
to initiate a bloom (Anderson, White, and Baden, 1985). In the Monterey
Bay, spring ocean conditions are markedly turbid. Due to a relatively high
nutrient level left over from turbulent mixing by the winter storms coupled
with an increasing light intensity with the onset of summer, this turbidity
is a result of increased phytoplankton growth. These conditions often lead
to visible red tides. In the spring of 1995 a Gymnodinium species developed
into a red tide in the Monterey Bay (personal correspondence with Deborah
Robertson). This happens to be a non-toxic species, so no special quarantines
were placed on shellfish this season (personal correspondence with the California
DHS).

When the bloom species is toxic, mussels, scallops, clams and other filter-feeding
bivalves all may be affected. DHS of California places a quarantine on potentially
toxic shellfish from May 1 through October 31, in addition to constant monitoring.
Worldwide monitoring programs are in effect, and in parts of Alaska
year-round bans of shellfish have been in effect since 1947. In Japan, red
tides devastate the pearl industry. Fish kills due to ciguatoxin -- a dinoflagellate
toxin that infects fish directly -- induce low yields, while bad publicity
due to PSP can affect fishing industries, even if the toxin has no direct
effect on the fish.

Ecologically, of course, dinoflagellates and blooms incur large effects
as well. As primary producers dinoflagellates must contribute to world CO2
consumption significantly, though not nearly as much as their numerous counterparts,
the diatoms. Diatoms grow much faster than dinoflagellates, however they
sink much faster. Hence, in turbid areas such as the temperate and polar
regions, where stirring holds the diatoms in the upper water column, they
dominate significantly over dinoflagellates. In the tropical seas, where
turbulence is at a minimum, dinoflagellates predominate in the plankton.
The slower growth rate of dinoflagellates, as well as a limited nutrient
base, contributes to the stereotypical clarity of tropical seas. Because
dinoflagellates use their flagella for motility they can vertically migrate
-- up during the day when the light is strongest, and down at night -- which
has consequences for the copepods who feed on them and on up the food chain.

Daily migration patterns have also been given significant attention because
of their association with circadian rhythms. In order for a behavior to
qualify as controlled by a circadian rhythm it must be shown in vitro to
occur without environmental cues. For example, dinoflagellates have been
shown to modify photosynthetic rate during the course of a day. A dinoflagellate
kept in sunlight for 24 hours will fix the most carbon towards the afternoon
and will fix much less carbon at "night," even though the light
intensity has not changed (Spector, 1984).

Because of their fast reproductive rate and their bioluminescence, dinoflagellates
make excellent subjects for circadian rhythm studies. At night glowing blue
flecks bounce off divers, and sailors have reported seeing the iridescent
outline of otherwise dark boats. The bioluminescence is produced by different,
fairly well studied biochemical reactions that produce visible light. The
chemicals involved in the reaction vary across species. Apparently, bioluminescence
has remarkably evolved many times, independently. These chemicals make excellent
biological tags for other biochemical experiments, making bioluminescent
dinoflagellates useful as model systems. Again it is tempting to ask why
something like bioluminescence may have evolved, and why so many times.
Today one can only speculate, for the true selection pressure remains elusive.

Perhaps the answer to evolutionary questions lies in certifying the phylogenetic
classification. Without a fossil record, the inferences made are strained
and derived, and unprovable. Suggestions have been made that dinoflagellates
may bridge the gap between prokaryotes and eukaryotes, may be much older
than the current fossil record reveals, may have evolved into brown algae,
or may represent their own unique evolutionary line. Other maybe's abound,
and now, two-hundred years since their discovery, much about the dinoflagellates
remains mysterious.